Triazole-fused sugars from nitroalkene-containing C-glycosides by a tandem 1,3-dipolar cycloaddition and intramolecular Michael addition

A synthetic method to triazole-fused sugars by treatment of nitroalkene-containing C-glycosides with sodium azide is described. Initial experiments conducted at room temperature gave only the 1,3-dipolar cycloaddition products. However, at elevated reaction temperature the tandem β-elimination/cycloaddition/Michael addition yielded 1,5-disubstituted triazole-fused sugars.     

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.Planetary protection efforts work toward protecting (i) solar system bodies from contamination by terrestrial biological material (forward contamination), thus preserving opportunities for future scientific investigation, and (ii) the Earth from harmful contamination by materials returned from outer space (back contamination) (5). These approaches apply directly to the control and eradication of microorganisms present on the surfaces of spacecraft intended to land, orbit, fly by, or be in the vicinity of extraterrestrial bodies. Consequently, current planetary protection policies require that spacecraft be assembled and readied for launch in controlled clean room environments. To achieve these conditions and maintain compliance with good manufacturing practice regulations, robotic spacecraft components are assembled in ultraclean facilities. Much like facilities in the medical, pharmaceutical, and semiconductor sectors, National Aeronautics and Space Administration (NASA) spacecraft assembly clean rooms (SAC) are kept extremely clean and are maintained to the highest of industry standards (17). Filtered air circulation, controlled temperature and humidity, routine exposure to disinfectants and surfactants, and nutrient-limiting, oligotrophic conditions make it very challenging for microorganisms to persist in such environments, but these measures by no means eradicate biological contaminants entirely (18). Several investigations, both culture based and culture independent, have demonstrated that a variety of bacterial taxa are repeatedly isolated under clean room conditions (18, 24, 26; P. Vaishampayan, S. Osman, G. Andersen, and K. Venkateswaran, submitted for publication). However, despite a growing understanding of the diverse microbial populations present in SAC, predicting the true risk of any such microbes’ compromising the findings of extraterrestrial life detection efforts remains a significant challenge (30). A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination (33).The purification of nucleic acids, subsequent PCR amplification, and shuttling of 16S ribosomal “fingerprint” genes from noncultivable microorganisms into genetically amenable lab strains of Escherichia coli have evolved into a gold standard of molecular means to elucidate the microbial diversity in a given sample. In theory, the cloning and sequencing of 16S ribosomal genes from each and every cell present, regardless of cultivability and inclusive of novel taxa, would result in a comprehensive survey of microbial communities on the surfaces of SAC and colocated spacecraft (24, 26). Unfortunately, the full-length sequencing of all 16S rRNA genes from environmental samples would be prohibitively expensive, making the approach unfeasible for generating comprehensive phylogenetic profiles of complex microbial communities.Attempting to infer population membership from clone libraries limited to hundreds or thousands of sequences has proven to be insufficient for detecting extremely low-abundance organisms. Recent analyses of phylogenetic DNA extracted from soil, water, and air revealed that laboriously derived clone libraries severely under-represent complex bacterial communities compared to very rapid (i.e., requiring only hours) DNA microarray approaches (1, 6, 11, 23, 36). One of the reasons for this is the high sensitivity of PhyloChip methodologies, which are able to detect organisms present in amounts below 10⁻⁴ abundance of the total sample (12). Numerous validation experiments using sequence-specific PCR have confirmed that taxa identified by the microarray were indeed present in the original environmental samples, despite their absence in corresponding clone libraries (3). This highlights the utility of the method compared to classical cloning. Although the analysis of each sample by the PhyloChip provides detailed information on microbial composition, the highly parallel and reproducible nature of this array allows tracking community dynamics over time and treatment. Even without prior sequence information, PhyloChip can identify specific microbial interactions that are key to particular changing environments.A comprehensive census of the microbial communities on the surfaces in three NASA SAC supporting two distinct missions was conducted. To ensure that the maximum diversity of resident microbiota was uncovered, subsamples from each clean room surface sampling were subjected to both DNA microarray protocols and conventional cloning and sequencing of 16S rRNA genes. This study, to our knowledge the first of its kind, focused on comparing the microbial diversity profiles resulting from DNA microarray analyses and conventional cloning and sequencing of 16S rRNA genes arising from a variety of low-biomass surfaces.     

Purification and biological characterization of a halophilic thermostable protease from <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> VKMM 007

An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures (20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0. Proteolytic activity was enhanced by Ca²⁺, K⁺, Mg²⁺, Na⁺, and Fe²⁺ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological potential of this novel halophilic protease.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Synthetic studies of neoclerodane diterpenes from Salvia divinorum: selective modification of the furan ring

A synthetic sequence has been developed to selectively functionalize the furan ring of the natural product salvinorin A (2a). The synthetic routes described convert the furan ring in 2a into an N-sulfonylpyrrole, oxazole or an oxadiazole. In addition, a procedure has been found to remove the furan skeleton completely. Biological results indicate that replacement of the furan ring with an N-sulfonylpyrrole leads to reduced affinity and efficacy at kappa opioid receptors.     

Substituent activity relationship studies on new azolo benzoxazepinyl oxazolidinones

In an effort to discover potent antibacterials based on the entropically favored 'bioactive conformation' approach, a series of novel tricyclic molecules mimicking the conformationally constrained structure of Linezolid is reported. Based on the initial tricyclic molecule 1, the benzazepine derivative 2 was designed where the tricyclic structure had more flexibility around C-N bond compared to 1. While, the molecule 2 was less active, the molecule 3 showed promising antibacterial activity presumably after having obtained rigidity due to pyrrole ring. The syntheses, SAR studies, and evaluation of 3 as a lead compound are reported.     

Familiarity with Own Population’s Appearance Influences Facial Preferences

Previous studies have found that individuals from rural areas in Malaysia and in El Salvador prefer heavier women than individuals from urban areas. Several explanations have been proposed to explain these differences in weight preferences but no study has explored familiarity as a possible explanation. We therefore sought to investigate participants’ face preferences while also examining the facial characteristics of the actual participants. Our results showed that participants from rural areas preferred heavier-looking female faces than participants from urban areas. We also found that the female faces from the rural areas were rated as looking heavier than the female faces from the urban areas. Our findings are consistent with the hypothesis that familiarity may be contributing to the differences found in face preferences between rural and urban areas given that people from rural and urban areas are exposed to different faces.     

Glutathione peroxidase3 of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3Δ cells during Cd-treatment.